Project 3 addresses two approaches to inducing human immune tolerance to the pig, namely mixed xenogeneic chimerism and porcine thymic transplantation, in a humanized (HU) mouse model. We have demonstrated that each approach can centrally tolerize human T cells to the donor pig and we hypothesize that the combination of both approaches will ultimately be optimal. During the current funding period, we have obtained evidence that mixed xenogeneic chimerism leads to specific tolerance or global unresponsiveness of human NK cells. We have obtained evidence that mixed xenogeneic chimerism tolerizes human T cell- independent B cells producing anti-pig xenoantibodies. We have shown that human T cells developing in human and porcine thymus grafts are functional and dependent on human APCs in the periphery for homeostasis. Human T cells developing in porcine thymus grafts, while tolerant of the donor pig, have reduced homeostatic expansion and reduced responses to antigen presented by HLA. We hypothesize that such abnormalities may be corrected while optimal protective immunity and tolerance to the pig and the human will be achieved by injecting autologous thymic epithelial cells (TECs) into the porcine thymic graft and combining transplantation of this ?hybrid? thymus with mixed xenogeneic chimerism. We will: 1) Engineer a pig/human ?hybrid? thymus to mediate positive and negative selection of human T cells on both pig and human MHC molecules. We will evaluate the impact of a pig/human TEC-containing hybrid thymus and of mixed xenogeneic chimerism on tolerance, homeostasis and function of human T cells. These studies will be directly relevant to the baboon studies in Project 1 and are expected to achieve optimal tolerance and protective immunity; and 2) Determine the effect of transgenic human CD47 expression on induction of pig chimerism in HU mice. We have shown that the lack of cross-species interaction between CD47 and SIRP? promotes macrophage-mediated rejection of xenogeneic hematopoietic cells. We have observed rapid destruction of porcine bone marrow cells by human immune systems in HU mice. We will determine the extent to which human CD47 expression prevents pig hematopoietic xenograft rejection by human and mouse macrophages in HU mice. Although transgenic expression of human CD47 inhibits pig cell destruction by human macrophages and prolongs pig chimerism in baboons, the hCD47 transgene still only allowed transient pig chimerism. Therefore, we will attempt to identify and overcome mechanisms of human rejection of hCD47- expressing pig bone marrow cells, which may reflect incomplete cross-reactivity of complement regulatory proteins (CRP). These studies will determine whether or not using hCRP transgenic pigs will further improve hCD47 Tg pig BMC survival. Additionally, we will test the impact of new genetic modifications of pigs from Project 4 on porcine chimerism in HU mice. Collectively, the studies will continue to inform the tolerance studies in Projects 1 and 2 and lead to the development of optimal transgenic pigs in Project 4.